The subject matter disclosed herein relates to acquisition and analysis of images of biological samples. More particularly, the disclosed subject matter relates to the calibration of microscopes used in such image acquisition protocols.
Certain types of molecular pathology examinations utilize a multiplexing workflow for molecular pathology imaging. When generating images using such a multiplexing workflow, a single slice of tissue (i.e., a single sample) may be used. The multiplexing workflow allows images of the tissue sample acquired over multiple rounds of imaging to be layered, with each round of imaging being directed to a different set of biomarkers applied to the sample; thus creating a composite view or image of the single sample. As such, the combination of biomarkers acquired over multiple rounds of imaging, displayed as a comprehensive view of tissue composition, provides for advanced analysis and diagnosis of the sample.
In certain workflows it would be advantageous to use different microscopes to obtain the images to be layered in creating the composite image. For a given lab with multiple microscopes, one problem arises with regard to load-balancing a fleet of microscopes performing multi-round imaging when, for example, one microscope is down for repair, or, alternatively, is backed up with many tissue samples that are tied to the microscope for the duration of the multi-round imaging process and are waiting for their additional rounds of imaging while other microscopes sit idle. Among the problems that may exacerbate management of multiple microscopes in such an arrangement is the need to calibrate (e.g., optically, geometrically, illumination, and so forth) all microscopes within a given lab relative to one another so that the layered images that are produced within a given lab are of high quality and are consistent over time, regardless of the microscope employed. To solve this problem, a comprehensive calibration process that enables microscope-independent imaging must be developed. Further, to minimize downtime of the microscopes and to maximize the throughput (the number of tissue slides imaged per day) in the lab, the calibration process should be as fast and efficient as possible.
When moving towards microscope-independent imaging, all microscopes need to be calibrated relative to each other through the use of a common reference standard to compensate for such factors as optical distortion, objective magnification, stage scale, stage rotation, stage offsets, and camera rotation. In some instances the reference standard could be a master microscope, while in other cases the reference standard could be an expected reference value for each calibration parameter from which offsets and ratios are computed relative to. By using a master microscope as the reference, the validity of the calibrations of all microscopes becomes vulnerable to changes or failures of the master microscope. When a change in the master microscope occurs, each microscope, including the master would need to be recalibrated. Relative traceability between the remaining microscopes could be obtained by designating a new master microscope to reinstate the traceability from. The problem with this approach is that any microscope could suffer a change or degradation in one or more properties at any time and therefore the validity of the inter-microscope calibrations could always be questioned.
Due to these reasons, the preferred approach is to use reference values or idealized models for each calibration parameter and calibrate each microscope relative to these parameters. The reference values do not change and therefore each microscope can be considered independently and calibrated independently to the standard. Periodic checks or routine automated calibrations can then be used to determine if any of the calibration parameters have deviated too far from their calibrated values and recalibrate if necessary.
Thus there remains a need for methods and devices to allow for accurate and reproducible inter-microscope calibration for registering images.